RT Journal Article SR Electronic T1 Assembly of Radically Recoded E. coli Genome Segments JF bioRxiv FD Cold Spring Harbor Laboratory SP 070417 DO 10.1101/070417 A1 Julie E. Norville A1 Cameron L. Gardner A1 Eduardo Aponte A1 Conor K. Camplisson A1 Alexandra Gonzales A1 David K. Barclay A1 Katerina A. Turner A1 Victoria Longe A1 Maria Mincheva A1 Jun Teramoto A1 Kento Tominaga A1 Ryota Sugimoto A1 James E. DiCarlo A1 Marc Guell A1 Eriona Hysolli A1 John Aach A1 Christopher J. Gregg A1 Barry L. Wanner A1 George M. Church YR 2016 UL http://biorxiv.org/content/early/2016/08/31/070417.abstract AB The large potential of radically recoded organisms (RROs) in medicine and industry depends on improved technologies for efficient assembly and testing of recoded genomes for biosafety and functionality. Here we describe a next generation platform for conjugative assembly genome engineering, termed CAGE 2.0, that enables the scarless integration of large synthetically recoded E. coli segments at isogenic and adjacent genomic loci. A stable tdk dual selective marker is employed to facilitate cyclical assembly and removal of attachment sites used for targeted segment delivery by sitespecific recombination. Bypassing the need for vector transformation harnesses the multi Mb capacity of CAGE, while minimizing artifacts associated with RecA-mediated homologous recombination. Our method expands the genome engineering toolkit for radical modification across many organisms and recombinase-mediated cassette exchange (RMCE).